Tetrahydroquinoline Derivatives and Their Use as Epac Inhibitors

ABSTRACT

The invention relates to tetrahydroquinoline derivatives and their use in the treatment and/or the prevention of a disease wherein the Epac protein is involved, such as inflammation, cancer, vascular diseases, kidney diseases, cognitive disorders and cardiac diseases.

The invention relates to tetrahydroquinoline derivatives, their use asEpac inhibitors and their pharmaceutical uses.

Cyclic adenosine 3′,5′-monophosphate (cAMP) is a universal secondmessenger that plays a crucial role in the intracellular signaltransduction of various stimuli controlling a wide variety of cellularevents including secretion, cell proliferation and differentiation,migration, and apoptosis. The guanine exchange factor (GEF) Epac(Exchange Protein directly Activated by Cyclic AMP) has been shown tocontribute to cAMP signalling in many processes (M. Breckler et al.,Rap-linked cAMP signaling Epac proteins: compartimentation, functioningand disease implications, Cell. Signal. 23 (2011) 1257-1266).

There are two isoforms of Epac, Epac1 and Epac2, both consisting of aregulatory region binding directly cAMP and a catalytic region thatpromotes the exchange of GDP (Guanosine diphosphate) for GTP(Guanosine-5′-triphosphate) on the Ras-like small GTPases Rap1 and Rap2isoforms. In the absence of cAMP, the regulatory region containing thecAMP-binding domain directly interacts with the catalytic region andinhibits its GEF activity. Binding of cAMP to Epac induces largeconformational changes within the protein and releases theautoinhibitory effect of the N-terminal region, leading to Rapactivation.

The two isoforms of Epac differ in that Epac1 has a single cyclicnucleotide-binding (CNB) domain, whereas Epac2 has two CNB domains,called CNB-A and CNB-B, which are located on both sides of the DEPdomain (Dishevelled, Egl-10 and Pleckstrin domain). The additionalN-terminal CNB domain in Epac2 has a low affinity for cAMP, and itsdeletion does not affect the regulation of Epac2 in response to agonists(J. de Rooij, H. Rehmann, M. van Triest, et al., Mechanism of regulationof the Epac family of cAMP-dependent RapGEFs, J. Biol. Chem. 275 (2000)20829-20836).

Epac plays critical roles in various physiological andpathophysiological processes such as memory formation, inflammation andcardiac remodelling (J. M. Enserink, et al., A novel Epac-specific cAMPanalog demonstrates independent regulation of Rap1 and ERK, Nat. CellBiol. 4 (2002) 901-906; Breckler et al., 2011). Therefore, there is aneed to provide Epac antagonists. There is also a need to providecompounds that inhibit downstream effectors such as Rap 1 and Rasfollowing the activation of Epac by Epac agonists. There is also a needto provide Epac antagonists that can be used for the prevention and/orthe treatment of diseases where Epac is involved such as inflammation,cancer, vascular diseases, kidney diseases, cognitive diseases andcardiac diseases.

The aim of the invention is to provide novel Epac antagonists.

Another aim of the invention is to provide novel Epac antagonists thatinhibit downstream effectors of Epac, even when Epac has been previouslyactivated by agonists.

Another aim of the present invention is to provide novel Epacantagonists which can be useful for the prevention and/or the treatmentof inflammation, cancer, vascular diseases, kidney diseases, cognitivediseases and cardiac diseases.

The present invention thus relates to a compound having formula (I):

wherein:

-   -   R9 is H or

is the attachment to the nitrogen atom of the tetrahydroquinoline;

-   -   R1, R2, R3, R4 and R8 are independently chosen from the group        consisting of:        H, (C₁-C₁₀)alkyl, (C₃-C₁₀)cycloalkyl, (C₆-C₁₀)aryl,        (C₁-C₆)alkylene-(C₆-C₁₀)aryl and (C₃-C₁₀)heteroaryl; said aryl        and heteroaryl groups being possibly substituted by at least one        substituent chosen from OH, NH₂, NO₂, (C₁-C₆)alkyl and halogen;    -   R5 is an halogen atom;    -   R6 and R7 are independently chosen from the group consisting of        H and halogen atoms;

or its pharmaceutically acceptable salts, hydrates or hydrated salts orits polymorphic crystalline structures, racemates, diastereomers orenantiomers,

for its use in the treatment and/or the prevention of a disease whereinthe Epac protein is involved.

The inventors have surprisingly discovered that tetrahydroquinolinederivatives inhibit the Epac protein. This inhibition leads to theinhibition of the Epac-induced Rap1 and Ras activation. Even moresurprising, tetrahydroquinoline derivatives inhibit Epac downstreameffectors Rap1 and Ras following Epac activation by Epac agonists.

Moreover, it has been shown that these tetrahydroquinoline derivativesare pharmacological inhibitors of Epac biological function, as theyblock an Epac1-dependent biological process, the cardiac myocytehypertrophy signalling. Previous studies demonstrated that, in responseto a prolonged β-AR (beta adrenergic receptors) stimulation, Epacinduced cardiac myocyte hypertrophy (Morel E; et al., cAMP-bindingprotein Epac induces cardiomyocyte hypertrophy Circ Res. 2005 Dec. 9;97(12):1296-304. Epub 2005 Nov. 3; Métrich M. et al., Epac mediatesbeta-adrenergic receptor-induced cardiomyocyte hypertrophy. Circ Res.2008 Apr. 25; 102(8):959-65. doi: 10.1161/CIRCRESAHA.107.164947. Epub2008 Mar. 6.).

In one embodiment, the compounds of formula (I) are Epac1 inhibitors. Inanother embodiment, the compounds of formula (I) are Epac1 selectiveinhibitors. In one embodiment, Epac1 selective inhibitors are compoundswhich exhibit an inhibitory effect on the Epac1 isoform. Moreparticularly, they generally exhibit an inhibitory effect on Epac1 andmoderate or no inhibitory effect on Epac2 isoform.

By “selective Epac1 inhibitor” it may be understood the ability of theEpac1 inhibitors to affect the particular Epac1 isoform, in preferenceto the other isoform Epac2. The Epac1 selective inhibitors may have theability to discriminate between these two isoforms, and so affectessentially the Epac1 isoform.

In one embodiment, they may exhibit a ratio of inhibition of Epac1versus Epac2 of at least 10 folds. In one embodiment, the compounds offormula (I) do not inhibit the protein kinase A (also called PKA). Inone embodiment, the compounds of formula (I) are not AMPc competitiveinhibitors.

The term “inhibitor” is to be understood as “antagonist”.

By “a disease wherein the Epac protein is involved” is meant a diseasewherein the Epac protein is expressed or over-expressed, and/or mutated.

The term “(C₁-C₁₀)alkyl” means a saturated or unsaturated aliphatichydrocarbon group which may be straight or branched having 1 to 10carbon atoms in the chain. Preferred alkyl groups have 1 to 4 carbonatoms in the chain, preferred alkyl groups are in particular methyl orethyl groups. “Branched” means that one or lower alkyl groups such asmethyl, ethyl or propyl are attached to a linear alkyl chain.

The term “(C₁-C₆)alkylene-” means a saturated or unsaturated aliphatichydrocarbon divalent radical which may be straight or branched having 1to 6 carbon atoms in the chain. For example, a preferred(C₁-C₆)alkylene-(C₆-C₁₀)aryl is a benzyl group.

By “(C₃-C₁₀)cycloalkyl” is meant a cyclic, saturated hydrocarbon grouphaving 3 to 10 carbon atoms, in particular cyclopropyl or cyclohexylgroups.

The term “(C₆-C₁₀)aryl” refers to an aromatic monocyclic, bicyclic, ortricyclic hydrocarbon ring system wherein any ring atom capable ofsubstitution may be substituted by a substituent. Examples of arylmoieties include, but are not limited to, phenyl.

The term “(C₃-C₁₀)heteroaryl” refers to an aromatic monocyclic,bicyclic, or tricyclic hydrocarbon ring system, wherein any ring atomcapable of substitution may be substituted by a substituent and whereinone or more carbon atom(s) are replaced by one or more heteroatom(s)such as nitrogen atom(s), oxygen atom(s) and sulphide atom(s); forexample 1 or 2 nitrogen atom(s), 1 or 2 oxygen atom(s), 1 or 2 sulphideatom(s) or a combination of different heteroatoms such as 1 nitrogenatom and 1 oxygen atom. Preferred heteroaryl groups are pyridyl,pyrimydyl and oxazyl groups.

The term “halogen” refers to the atoms of the group 17 of the periodictable and includes in particular fluorine, chlorine, bromine, and iodineatoms, more preferably fluorine, chlorine and bromine atoms.

By “tetrahydroquinoline” it is understood the following group:

The compounds herein described may have asymmetric centers. Compounds ofthe present invention containing an asymmetrically substituted atom maybe isolated in optically active or racemic forms. It is well-known inthe art how to prepare optically active forms, such as by resolution ofracemic forms or by synthesis from optically active starting materials.All chiral, diastereomeric, racemic forms and all geometric isomericforms of a compound are intended, unless the stereochemistry or theisomeric form is specifically indicated. In an embodiment, the carbonatom referred to with (*) in the formula (I) with R2 to R9 as definedabove may be (R) or (S):

In an embodiment it is (R). In a particular embodiment, the enantiomericform (R) of the compound of formula (I) is preferred and moreparticularly the following enantiomeric form:

In another embodiment, the (R)-enantiomeric form of the compound offormula (I) is a more potent cAMP antagonist than racemic and(S)-enantiomeric form of the compound of formula (I). In one embodiment,the (R)-enantiomeric form of the compound of formula (I) is a selectiveinhibitor of Epac1. Said (R)-enantiomeric form may inhibit the GEFactivity of Epac1 with 10-times more efficiency than the(S)-enantiomeric form.

The term “pharmaceutically acceptable salt” refers to salts which retainthe biological effectiveness and properties of the compounds of theinvention and which are not biologically or otherwise undesirable.Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids, while pharmaceutically acceptable baseaddition salts can be prepared from inorganic and organic bases. For areview of pharmaceutically acceptable salts see Berge, et al. ((1977) J.Pharm. Sd, vol. 66, 1). For example, the salts include those derivedfrom inorganic acids such as hydrochloric, hydrobromic, sulfuric,sulfamic, phosphoric, nitric, and the like, as well as salts preparedfrom organic acids such as acetic, propionic, succinic, glycolic,stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic,hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,fumaric, methanesulfonic, and toluenesulfonic acid and the like.

In a particular embodiment, the compounds of the invention have thefollowing formula:

that is in formula (I), R9 is

In a particular embodiment, the compounds of the invention have thefollowing formula (II):

wherein

-   -   R1, R2, R3, R4 and R8 are independently chosen from the group        consisting of:        H, (C₁-C₁₀)alkyl, (C₃-C₁₀)cycloalkyl, (C₆-C₁₀)aryl,        (C₁-C₆)alkylene-(C₆-C₁₀)aryl and (C₃-C₁₀)heteroaryl; said aryl        and heteroaryl groups being possibly substituted by at least one        substituent chosen from OH, NH₂, NO₂, (C₁-C₆)alkyl and halogen;    -   R5 is an halogen atom;    -   R6 and R7 are independently chosen from the group consisting of        H and halogen atoms;        or its pharmaceutically acceptable salts, hydrates or hydrated        salts or its polymorphic crystalline structures, racemates,        diastereomers or enantiomers,        for its use in the treatment and/or the prevention of a disease        wherein the Epac protein is involved.

In a particular embodiment, in formula (II) as defined above R1 is H.

The above embodiments refer either to formula (I) or to formula (II).

In a particular embodiment, R2 is H or a (C₁-C₁₀)alkyl.

In a particular embodiment, R2 is a (C₁-C₁₀)alkyl. Preferably, R2 is a(C₁-C₄)alkyl.

More preferably, R2 is a methyl group. In another embodiment, R2 is H.Preferably, R2 is H or a methyl group.

In a particular embodiment, R3 is H.

In another embodiment, R4 is H.

In another embodiment, R8 is H.

In one embodiment, R3, R4 and R8 are H.

In a particular embodiment, the (C₃-C₁₀)heteroaryl group is chosen fromthe group consisting of pyridyl, pyrimydyl and oxazyl groups.

In another embodiment, the (C₆-C₁₀)aryl group is a phenyl group.

In another embodiment, the (C₁-C₆)alkylene-(C₆-C₁₀)aryl is a benzylgroup.

In a particular embodiment, R5 is chosen from the group consisting of F,Cl, Br and I. Preferably, R5 is Br.

In a particular embodiment, R6 is chosen from the group consisting of H,F, Cl, Br and I. In a particular embodiment, R6 is chosen from the groupconsisting of F, Cl, Br and I. In another embodiment, R6 is F. Inanother embodiment R6 is H. Preferably, R6 is H or F.

In a particular embodiment, R7 is chosen from the group consisting of H,F, Cl, Br and I. In a particular embodiment, R7 is chosen from the groupconsisting of F, Cl, Br and I. In another embodiment, R7 is Br. Inanother embodiment, R7 is H. Preferably, R7 is H or Br.

In a preferred embodiment, R1 is H and R5 is Br.

In another preferred embodiment, at least two of R5, R6 and R7 arehalogen.

The above mentioned particular embodiments can be combined with eachother. Some specific compounds for the use as defined above have thefollowing formulae:

named herein CE3F4.

More particularly, some specific compounds for the use as defined abovehave the following formulae:

In one embodiment, the compound of formula (I) is:

The present application also describes the following compounds:

The invention also relates to a compound having one of the followingformulae:

Preparation of the Compounds of Formula (I):

The compounds of formula (I) can be synthesized according to previouslypublished methods in P. Bouyssou et al., J. Heterocyclic Chem., 29, 895,1992. Methods of preparation of the compounds of formula (I) arewell-known.

The present invention also relates to a pharmaceutical composition,comprising a compound having formula (I) for its use as defined above,in association with at least one pharmaceutically acceptable excipient.

The present invention also relates to a drug, comprising a compoundhaving formula (I) for its use as defined above.

While it is possible for the compounds having formula (I) to beadministered alone, it is preferred to present them as pharmaceuticalcompositions. The pharmaceutical compositions, both for veterinary andfor human use, useful according to the present invention comprise atleast one compound having formula (I) as above defined, together withone or more pharmaceutically acceptable carriers and possibly othertherapeutic ingredients.

In certain preferred embodiments, active ingredients necessary incombination therapy may be combined in a single pharmaceuticalcomposition for simultaneous administration.

As used herein, the term “pharmaceutically acceptable” and grammaticalvariations thereof, as they refer to compositions, carriers, diluentsand reagents, are used interchangeably and represent that the materialsare capable of administration to or upon a mammal without the productionof undesirable physiological effects such as nausea, dizziness, gastricupset and the like.

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well understood in the artand need not be limited based on formulation. Typically suchcompositions are prepared as injectables either as liquid solutions orsuspensions; however, solid forms suitable for solution, or suspensions,in liquid prior to use can also be prepared. The preparation can also beemulsified. In particular, the pharmaceutical compositions may beformulated in solid dosage form, for example capsules, tablets, pills,powders, dragees or granules.

The choice of vehicle and the content of active substance in the vehicleare generally determined in accordance with the solubility and chemicalproperties of the active compound, the particular mode of administrationand the provisions to be observed in pharmaceutical practice. Forexample, excipients such as lactose, sodium citrate, calcium carbonate,dicalcium phosphate and disintegrating agents such as starch, alginicacids and certain complex silicates combined with lubricants such asmagnesium stearate, sodium lauryl sulphate and talc may be used forpreparing tablets. To prepare a capsule, it is advantageous to uselactose and high molecular weight polyethylene glycols. When aqueoussuspensions are used they can contain emulsifying agents or agents whichfacilitate suspension. Diluents such as sucrose, ethanol, polyethyleneglycol, propylene glycol, glycerol and chloroform or mixtures thereofmay also be used.

The pharmaceutical compositions can be administered in a suitableformulation to humans and animals by topical or systemic administration,including oral, rectal, nasal, buccal, ocular, sublingual, transdermal,rectal, topical, vaginal, parenteral (including subcutaneous,intra-arterial, intramuscular, intravenous, intradermal, intrathecal andepidural), intracisternal and intraperitoneal. It will be appreciatedthat the preferred route may vary with for example the condition of therecipient.

The formulations can be prepared in unit dosage form by any of themethods well known in the art of pharmacy. Such methods include the stepof bringing into association the active ingredient with the carrierwhich constitutes one or more accessory ingredients. In general theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then, if necessary, shaping the product.

The invention relates to a compound having formula (I) as defined above,for its use for the treatment and/or the prevention of inflammation,cancer, vascular diseases including atherogenesis, atherosclerosis andpostangioplasty restenosis, kidney diseases including diabeticnephropathy, cognitive disorders and cardiac diseases.

The invention also relates to the use of a compound having formula (I)as defined above, for the preparation of a medicament for the treatmentand/or the prevention of inflammation, cancer, vascular diseasesincluding atherogenesis, atherosclerosis and postangioplasty restenosis,kidney diseases including diabetic nephropathy, cognitive disorders andcardiac diseases.

By “inflammation” is meant phenomena by which the human body defendsitself against aggression and which can manifest itself in varioussymptoms such as pain, swelling, heat or redness of the skin.

By the term “cancer” is meant solid tumors and/or disseminatedhematological cancers and/or their metastasis. The terms “metastasis” or“metastatic diseases” refer to secondary tumors that are formed by cellsfrom a primary tumor which have moved to another localization. The term“hematological cancers” refers to types of cancer that affect blood,bone marrow, and lymph nodes such as myelomas, lymphomas or leukemias.

Cognitive disorders are a category of mental health disorders thatprimarily affect learning, memory, perception, and problem solving, andinclude amnesia, dementia, and delirium, more particularly Alzheimer'sdisease.

Cardiac diseases more specifically point out cardiac hypertrophy,cardiac arrhythmias, valvulopathies, diastolic dysfunction, chronicheart failure, ischemic heart failure, myocarditis, hypertrophic anddilated cardiomyopathies. It has to be noted that besides Rap1, Epac1has been shown to activate the small GTPase H-Ras in different celltypes including primary cardiomyocytes (Keiper et al., 2004; Métrich etal., 2008; Métrich et al., 2010a, 2010b; Schmidt et al., 2001).

The invention thus also relates to a compound of formula (I) as definedabove, for its use for the treatment and/or the prevention of cardiacdiseases which are selected from the group consisting of cardiachypertrophy, cardiac arrhythmias, valvulopathies, diastolic dysfunction,chronic heart failure, ischemic heart failure, myocarditis, hypertrophicand dilated cardiomyopathies, preferably cardiac hypertrophy.

The invention also relates to a method of prevention and/or treatment ofa disease wherein the Epac protein is involved, said method comprisingthe administration of a pharmaceutical acceptable amount of a compoundof formula (I) as defined above to a patient in need thereof.Preferably, the present invention relates to a method of preventionand/or treatment of inflammation, cancer, vascular diseases includingatherogenesis, atherosclerosis and postangioplasty restenosis, kidneydiseases including diabetic nephropathy, cognitive diseases and cardiacdiseases, said method comprising the administration of a pharmaceuticalacceptable amount of a compound of formula (I) as defined above to apatient in need thereof.

More particularly, the invention relates to a method of preventionand/or treatment of cardiac hypertrophy, cardiac arrhythmias,valvulopathies, diastolic dysfunction, chronic heart failure, ischemicheart failure, myocarditis, hypertrophic and dilated cardiomyopathies,said method comprising the administration of a pharmaceutical acceptableamount of a compound of formula (I) as defined above to a patient inneed thereof.

In the context of the invention, the term “treating” or “treatment”, asused herein, means reversing, alleviating, inhibiting the progress of,or preventing the disorder or condition to which such term applies, orone or more symptoms of such disorder or condition.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the inhibitory activity of CE3F4 on the exchange activityof Epac1. Epac1 nucleotide exchange activity was measured as a functionof time (in seconds) in the absence of compound 007 (curve with squares)or in the presence of 2 μM of compound 007, either alone (curve withclosed circles) or with 25 μM of compound 009 (curve with triangles) orwith 20 μM of CE3F4 (curve with open circles).

FIGS. 2A-C. Inhibitory activity of CE3F4 on Epac-induced Rap1activation. (A) HEK293 cells transfected with either pcDNA3 controlvector or Epac1; (B) HEK293 cells transfected with Epac1 and 24 h aftertransfection preincubated or not with CE3F4 and then treated or not withSp-007; (C) HEK293 cells overexpressing β1AR (Beta-1 adrenergicreceptors) and transfected with Epac1 pretreated or not with CE3F4 andstimulated or not with Iso.

FIGS. 3A-C. Inhibitory activity of CE3F4 on Epac-induced Ras activation.

FIG. 3A shows the inhibitory activity of CE3F4 on Epac-induced Rasactivation in HEK293 cells.

FIG. 3B shows the inhibitory activity of CE3F4 on Epac-induced Rasactivation in rat neonatal cardiac myocytes.

FIG. 3C shows the inhibitory activity of CE3F4 on Epac-induced Rasactivation in HEK293 cells overexpressing β-AR.

FIGS. 4A and 4B show that CE3F4 prevents Epac-induced cardiacremodeling.

FIG. 4A shows the inhibition of the Iso-induced ANF-Luc transcriptionalactivation by CE3F4.

FIG. 4B shows the inhibition of the Iso-induced NFAT-Luc transcriptionalactivation by CE3F4.

FIG. 5 shows initial velocities of GEF activity of Epac1 activated by 20μM of 007 in the presence of increasing concentrations of racemic CE3F4(empty circles), (R)-CE3F4 (black squares), (S)-CE3F4 (black diamonds),and compound f) (empty triangles).

FIGS. 6A-C show the initial velocity of GDP exchange catalyzed by Epac1,Epac2(AB), or Epac2(B), in the presence or absence of agonists and ofinhibitory compounds. The agonist-dependent activity was obtained bysubtraction of the constitutive GDP exchange activity, measured in thepresence of Epac proteins but in the absence of agonists and inhibitors.

FIG. 6A shows the initial velocity of GDP exchange catalyzed by Epac1(black bars), Epac2(AB) (empty bars), and Epac2(B) (hatched bars)incubated with 50 μM racemic CE3F4 and with 50 μM 007 or 300 μM cAMP, asindicated below the x-axis.

FIG. 6B shows Epac1 (black circles) or Epac2B (empty squares) activatedby 300 μM cAMP, and the initial velocity of GDP exchange in the presenceof increasing concentrations of (R)-CE3F4.

FIG. 6C shows the initial velocity of GDP exchange catalyzed by Epac1(hatched bars) or Epac2(B) (black bars) measured in the presence of 30μM racemic CE3F4 or compounds a) from h), and 300 μM cAMP as theagonist. Results are expressed relative to the control values measuredin the absence of inhibitor, which were set at 100%. p<0.01% for globalcomparison of Epac2(B) and Epac1 (Wilcoxon matched-pair signed ranktest). p<5% or less for comparison of Epac2(B) and Epac1 to be inhibitedby each compound (one-tailed Student's t-test).

The following examples represent some specific embodiments of theinvention and can not be considered as limiting.

EXAMPLES

In the following examples:

-   -   “CE3F4” refers to the specific compound having the following        formula:

-   -   -   CE3F4 and related compounds a), b), c), d), g), and h) were            synthesized according to methods described in P. Bouyssou,            et al., Synthesis of 7- and            5,7-substituted-6-fluoro-2-methyl-1,2,3,4-tetrahydroquinolines:            convenient precursors of quinolone antibacterial agents, J.            Heterocyclic Chem. 29 (1992) 895-898.        -   compound f) was synthesized from 6-fluoroquinoline by            reduction and formylation of this compound to give            6-fluoro-1-formyl-1,2,3,4-tetrahydroquinoline, which was            then brominated to give compound f).        -   compound e) was synthesized from 3,5-dibromoaniline by a            Skraup synthesis to give 5,7-dibromoquinaldine followed by            reduction of nitrogen-containing ring and formylation of            nitrogen in position 1.        -   The (R)- and (S)-enantiomers of CE3F4 were derived from the            (R)- and (S)-enantiomers of            6-fluoro-2-methyl-1,2,3,4-tetrahydroquinoline (6-FTHQ). (R)-            and (S)-6-FTHQ were obtained according to methods described            in J. F. Gerster, et al., Synthesis, absolute configuration,            and antibacterial activity of            6,7-dihydro-5,8-dimethyl-9-fluoro-1-oxo-1H,5H-benzo[ij]quinolizine-2-carboxylic            acid, J. Med. Chem. 30 (1987) 839-843 and were then            formylated and brominated to give, respectively, (R)-CE3F4            and (S)-CE3F4.

    -   The mass spectra and nuclear magnetic resonance of these        compounds were identical to those reported previously for        racemic CE3F4 (Bouyssou P., 1990. Contribution à ĺetude des        quinolones carboxyliques de structure type Benzo [i,j]        Quinolizine, relation structure activité. Ph.D. Thesis. Orleans        University, France). Optical rotation measurements were        performed at 589 nm, 20° C. and c=10 mg/ml in chloroform, with        an accuracy of ±1°, using a Perkin Elmer model 341 polarimeter.        (R)-CE3F4 is levorotatory with [α]D=−12±1°, while (S)-CE3F4 is        dextrorotatory with [α]D=+11±1°.

    -   “Compound 007” refers to the Epac agonist,        8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic        monophosphate, obtained from Biolog, Bremen, Germany. “Compound        007”, is a cAMP analog which activates Epac, but not PKA.

    -   “Compound 009” refers to        8-(4-chlorophenylthio)-guanosine-3′,5′-cyclic monophosphate,        obtained from Biolog, Bremen, Germany.

    -   “Sp-007” refers to a membrane-permeant Epac agonist,        Sp-8-pCPT-2′-O-Me-cAMPS (Christensen et al., 2003).

    -   Isoprenaline (also called “Iso”) was obtained from        Sigma-Aldrich.

    -   BODIPY FL 2′-(or-3′)-O—(N-(2-aminoethyl)urethane),        bis(triethylammonium) salt (Bodipy-GDP) was obtained from        Invitrogen.

Example 1: Kinetic Characteristics of Epac Inhibition by CE3F4

The effects of CE3F4 on the exchange activity of Epac-1 were studied asfollows.

Protocol:

Epac1 nucleotide exchange activity was measured:

-   -   in the absence of compound 007,    -   or in the presence of 2 μM of compound 007, either alone or with        25 μM of compound 009,    -   or with 20 μM of CE3F4.

Variations of RFU (Relative Fluorescence Units) were studied as afunction of time and fitted to single exponentials. Reported values aremean±SEM (n=3). (see Materiel and Methods).

Results:

FIG. 1 shows that a single dose of CE3F4 inhibited the exchange reactioninduced by Epac1 plus 007 (2 μM) to an extent similar to the inhibitoryeffect of 009 at 25 μM.

Example 2: Effects of CE3F4 on Epac Downstream Effectors in CulturedCells

1) Effects of CE3F4 on Epac1-Induced Rap1 Activation:

The ability of CE3F4 to block Epac1-induced Rap1 activation in culturedHEK293 cells was tested according to the following experiment.

Protocol:

HEK293 cells were transfected with either pcDNA3 control vector or Epac1(see FIG. 2A).

HEK293 cells were transfected with Epac1 and 24 h after transfection,cells were preincubated or not with CE3F4 for 30 min and were thentreated or not with Sp-007 (10 μM) for 10 min (see FIG. 2B).

HEK293 cells overexpressing β1AR (Beta-1 adrenergic receptors) andtransfected with Epac1 were pretreated or not with CE3F4 and stimulatedor not with Iso (10 μM) for 10 min (see FIG. 2C).

Amounts of Rap1-GTP were determined by pull-down assays (see Materialand Methods). The bar graph represents the mean±S.E.M. of 5 (FIG. 2A,2B) or 3 (FIG. 2C) independent experiments. Results are expressed as thepercentage of unstimulated control cells.

Results:

FIG. 2A shows that with the vector control, even in the presence ofSp-007, the ratio Rap1-GTP/Total Rap1 is lower than for cellstransfected with Epac1, and that in presence of Sp-700, the cellstransfected with Epac1 had higher level of Rap1-GTP. Sp-007 induced arobust activation of Rap1 in cells overexpressing Epac1 compared tocontrol cells transfected with the empty vector.

CE3F4 (20 μM) prevented the increase in the amount of Rap1-GTP following10 μM of Sp-007 treatment (FIG. 2B). Similarly, CE3F4 decreasedEpac1-induced Rap1 activation following stimulation of β₁-adrenergicreceptor (β₁-AR) by isoprenaline (Iso, 10 μM) (FIG. 2C).

2) Effects of CE3F4 on Epac1-Induced H-Ras Activation:

The effects of CE3F4 to prevent Epac1-induced H-Ras activation wasdetermined by extraction of GTP-loaded H-Ras from cell lysates with theimmobilized Ras-binding domain of Raf1.

Protocol:

HEK293 cells (FIG. 3A) or rat neonatal cardiac myocytes (FIG. 3B) weretransfected with Epac1. 24 h after transfection, cells were preincubatedor not with CE3F4 for 30 min and were then treated or not with Sp-007(10 μM) for 10 min. In FIG. 3C, HEK293 cells overexpressing β₁-AR andtransfected with Epac1 were pretreated or not with 20 μM of CE3F4 for 30min and stimulated or not with Iso for 10 min. Amounts of H-Ras-GTP weredetermined by pull-down assays. The bar graph represents the mean±S.E.M.of 5 (FIG. 3A) or 2 (FIG. 3B, 3C) independent experiments. Results areexpressed as the percentage of unstimulated control cells.

Results:

As observed for Rap1, the amount of Ras-GTP induced by either Sp-007 orIso was decreased in the presence of CE3F4 in HEK293 and primary ratcardiac myocytes (FIGS. 3A, 3B and 3C).

Conclusion

Altogether these data show that CE3F4 is efficient in preventingEpac-induced Rap1 and Ras activation in cultured cells.

Example 3: CE3F4 Prevents Epac-Induced Cardiac Myocyte Hypertrophy

The potential biological effects of CE3F4 on Epac-induced cardiacmyocyte growth following β-AR stimulation were investigated as follows.

ANF (Atrial Natriuretic Factor) is a marker of myocyte hypertrophy.NFAT is a nuclear factor involved in Epac pro-hypertrophic signalling(see Métrich et al., 2010b).ANF and NFAT promoters were fused to the Firefly luciferase gene(ANF-Luc) (NFAT-Luc) to detect ANF and NFAT by Firefly Luciferase assay(see Materiel and Methods).

Protocol:

A) Iso-Induced ANF-Luc Transcriptional Activation is Blocked by CE3F4:

Rat neonatal cardiomyocytes were cotransfected with ANF-Luc and Epac1 orempty vector pcDNA3 as control. One day after transfection, cells werepretreated or not with CE3F4 (20 μM) for 30 min and stimulated or notwith Iso (10 μM) for 8 h. Cells were then assayed for Luciferaseactivity.

B) Iso-Induced NFAT-Luc Transcriptional Activation is Blocked by CE3F4:

Neonatal cardiomyocytes were transfected with NFAT-Luc. One day latercells were pretreated or not with CE3F4 (20 μM) for 30 min andstimulated or not with Iso (10 μM) for 8 h. The day after, cells wereassayed for Luciferase activity.

Results were normalized to control for each experiment, and wereexpressed as means±S.E.M of at 4 (FIG. 4A) or 5 (FIG. 4B) independentexperiments performed in triplicates.

Results:

Iso increased ANF as shown in FIG. 4A. The effect of Iso was boosted incardiomyoctes overexpressing Epac (FIG. 4A). Importantly, treatment ofcardiac myocytes with CE3F4 significantly inhibited Iso-induced ANF-Lucgene transcriptional activity (FIG. 4A).

Consistent with this finding, CE3F4 prevented Iso-induced NFATtranscriptional activity (FIG. 4B).

Conclusion:

These data indicate that CE3F4 is a pharmacological inhibitor of Epacbiological function.

Example 4: (R)-CE3F4 is a More Potent Enantiomer that Antagonizes Epac1Activation by cAMP than the (S)-CE3F4

FIG. 5 shows a representative dose-response inhibition assay in whichthe GEF activity of 007-activated Epac1 toward Rap1 was measured in thepresence of increasing concentrations of racemic CE3F4 or of itsindividual enantiomers (R)-(−)-CE3F4 and (S)-(+)-CE3F4. Thedose-response curve for the (R)-enantiomer is left-shifted relative tothat obtained with the racemate and with the (S)-enantiomer, showingthat the (R)-(−)-CE3F4 is a more potent Epac inhibitor.

The compound f) was also tested: the dose-response curve was notsignificantly different (F test) from the one obtained with (S)-CE3F4.Each experimental point is the mean of initial velocity values computedfrom triplicate time-course experiments.

The IC₅₀ values for the racemate, enantiomers and compound f) weredetermined several times independently:

Mean EC50 (μm) SD n p CE3F4 10.7 1.4 6 <1% (R)-CE3F4 5.8 0.8 9 <1%(S)-CE3F4 56 7 8 NS Compound f) 50 5 4 NS

(R)-CE3F4 inhibited Epac1 GEF activity with an IC₅₀ which was about2-fold smaller than that obtained with the racemic CE3F4, and about10-fold smaller that the IC₅₀ values of (S)-CE3F4 and compound f). IC₅₀values were computed using Graphpad Prism, according to afour-parameters dose-response model. (C) Mean±SD of n=4 to 9 independentdeterminations of IC₅₀ values for each inhibitor.

Example 5: (R)-CE3F4 and its Analogs Differentially Inhibit Epac1 andEpac2

Epac1, Epac2(AB) that possesses both the CNB-A and the CNB-B domains, orEpac2(B) that is deleted of its CNB-A domain, were activated bysaturating concentrations of either 007 (50 μM) or cAMP (300 μM).

FIG. 6A shows that, whatever the agonist used, the GEF activity of Epac1was much more inhibited by 50 μM of racemic CE3F4 than the GEF activityof either Epac2(AB) or Epac2(B). On the other hand, there was nosignificant difference in the inhibitory effects of racemic CE3F4 on theGEF activity of Epac2(AB) or of Epac2(B). (R)-CE3F4 was then used tostudy the concentration-dependent inhibition of the GEF activities ofEpac1 and Epac2(B) activated by cAMP (300 μM). Results are expressed asthe % of the initial velocity of GDP exchange measured in the absence ofracemic CE3F4. *, p<1%; ns (not significant), p>5% by two-tailedStudent's t-test.

FIG. 6B shows that the dose-response curve obtained with Epac2(B) isstrongly right-shifted relative to that obtained with Epac1. From twoindependent dose-response experiments such as the one shown in FIG. 6B,the mean IC₅₀ of (R)-CE3F4 was 4.2 μM for Epac1 and 44 μM for Epac2(B).Results are expressed as the % of the initial velocity of GDP exchangemeasured in the absence of (R)-CE3F4. p<0.1% by comparison of fits(IC₅₀) based of the extra sum-of square F test (Graphpad Prism).

FIG. 6C: cAMP-activated Epac1 and Epac2(B) were compared for theirability to be inhibited by several CE3F4 structural analogs. All of themare in the racemic form, except for the compound f). FIG. 6C shows theresults of a representative experiment, expressed as the relativeinhibitory potencies of the various analogs (30 μM), measured in thepresence of cAMP (300 μM) as the agonist.

Compounds a), e) and f) showed antagonistic properties toward both Epacisoforms even if they were weaker cAMP antagonists toward Epac2(B) thantoward Epac1 by two-tailed Wilcoxon matched-pair signed rank test (pvalue <1%,). Compounds g) and h) showed antagonistic properties towardEpac1.

The stronger antagonist of Epac1 and Epac 2(B) activation by cAMP wasCE3F4 itself. A dose-response study (not shown) indicated that the IC₅₀of racemic CE3F4 was 11 μM for Epac1 and 66 μM for Epac2(B) when theywere activated by 300 μM cAMP.

Comparative compounds b), c) and d), which are not encompassed by theformula (I) of the compounds of the invention, showed no significantinhibitory activity on both Epac isoforms.

Material and Methods:

Measurement of In Vitro Activation of Epac1

Method Used to Determine Epac1 Exchange Activity:

Concerning FIG. 1, a “multipoint” (time-course) method was used. Allcomponents except the agonist (007) were mixed in a well and theexchange activity of Epac1 was initiated by injection of the agonist.GST-Rapt was preloaded with a fluorescent derivative of GDP, and theEpac1-catalyzed nucleotide exchange was measured using a large excess ofnon-fluorescent GDP, by taking advantage of the spectroscopic differencebetween free and Rap1-bound fluorescently labeled GDP. The principle ofthe method is similar to that reported in particular by Van den Bergheet al. (1997; Oncogene 15, 845), except that Bodipy FL-labeled GDP(bGDP) was used here, rather than 2′,3′-bis(O)—N-methylanthraniloyl-GDP(mant-GDP).

To determine Epac1 exchange activity, 200 nM of purified GST-Rap1Apreloaded with bGDP were incubated at 22° C. in exchange buffer (50 mMTris-HCl, pH 7.5, 50 mM NaCl, 5 mM MgCl₂, 5 mM DTE, 5% glycerol, 0.01%NP40), in the presence of 100 nM of purified GST-Epac1, 20 μM ofunlabeled GDP, and defined concentrations of compound 007, compound 009and CE3F4. Experiments were performed in black 384-well plates (CorningInc. Ref 3573) in a final volume of 30 μL. bGDP fluorescence (Ex=480 nm;Em=535 nm) was measured using a multilabel plate reader (Envision Xcite,Perkin-Elmer).

Method used to measure the amount of either Rap1-GTP or Ras-GTP inducedby Epac: see pulldown assay.

Cell Culture:

Cardiac myocytes were isolated as previously described by Wollert andcolleagues (1996). HEK293 cells stably expressing the β₁-adrenergicreceptor (β₁-AR) was a gift of Dr. Shenoy (Duke University). HEK293cells were maintained in MEM with FBS (Foetal Bovine Serum; 10%) andpenicillin-streptomycin (1%). All media, sera and antibiotics used incell culture were purchased from Invitrogen (Cergy Pontoise, France).

Plasmid and Transfection:

The plasmid constructs were generously provided as following:

The rat ANF promoter fused to the Firefly luciferase reporter gene(ANF-Luc) by Dr K. Knowlton, Epac and Rap1 plasmid construct(Epac1^(WT)) by Drs J. L. Bos and J. de Gunzburg, respectively. TheFirefly luciferase reporter plasmid driven by four NFAT consensusbinding sites (NFAT-Luc) was purchased from Stratagene. Transienttransfection experiments of HEK cells and primary cardiac myocytes wereperformed with respectively X-treme GENE 9 reagent (Roche AppliedScience) and Lipofectamine 2000 (Invitrogen Life Technologies) in thepresence of various amounts of plasmid constructs according to themanufacturer's instructions.

Firefly Luciferase Assay:

Cells were lysed, and luminescence was detected using the LuciferaseAssay System (Promega) according to the manufacturer's instructions withTecan Infinite.

Pull-Down Assay:

Ras and Rap1 pull-down experiments were performed using a GST fusionprotein containing respectively the Ras binding domain of Raf1-RBD andthe Rap1 binding domain of Ral-GDS as previously described (Métrich etal., 2008). Cells were starved for 1 h before stimulation in MEM freewith penicillin-streptomycin (1%). After stimulation, cells were lysedin RIPA buffer (50 mM Tris-HCl, pH 7.5; 500 mM NaCl; 20 mM MgCl2; 0.5%deoxycholic acid; 0.1% SDS; 1% Triton X-100; 1 mM PMSF; protease andphosphatase inhibitors) and 500 μg of protein were incubated with eitherGST-Raf1-RBD (for Ras) or Ral-GDS (for Rap1) coupled toglutathione-Sepharose beads (Amersham Biosciences) for 1h at 4° C. Beadswere then washed three times in washing buffer (50 mM Tris-HCl, pH 7.5;150 mM NaCl, 20 mM MgCl₂, 1% Triton X-100; 0.1 mM PMSF; protease andphosphatase inhibitors). Rap1-GTP or Ras samples and corresponding totallysates were separated on SDS-PAGE gels and transferred onto apolyvinylidene difluoride (PVDF) membrane (Amersham Pharmacia Biotech).Membranes were revealed with Dura kit (Pierce).

Recombinant Protein Expression:

Protein NCBI Reference Sequences for human Epac1 and Epac2 are NP_006096and NP_008954, respectively. Recombinant human Epac1 (residues 149-881),deleted of its Dishevelled, Egl-10, and Pleckstrin (DEP) domain, andhuman Rap1A were produced with GST as a fusion tag (D. Courilleau etal., Identification of a tetrahydroquinoline analog as a pharmacologicalinhibitor of the cAMP-binding protein Epac, J. Biol. Chem. 287 (2012)44192-44202).

Human Epac2(AB) (amino acids 43-1011) carries both the CNB-A and theCNB-B domains. Human Epac2(B) (amino acids 283-1011) lacks the first 283amino acids of Epac2. It is therefore deleted of its CNB-A and DEPdomains, but retains the CNB-B domain and the full catalytic region.Epac2(AB) was obtained by HindIII/NotI restriction and Epac2(B) wasobtained by Ssp1/Not1 restriction of a human Epac2A cDNA (a gift fromAnn M Graybiel, Department of Brain and Cognitive Sciences, MIT,Cambridge). Both cDNA fragments were inserted into pET41a (Novagen),expressed in Escherichia coli Rosetta 2(DE3) (Novagen), and theGST-tagged fusion proteins were purified by nickel-nitrilotriacetic acidbeads (Qiagen), as described in D. Courilleau, et al., Identification ofa tetrahydroquinoline analog as a pharmacological inhibitor of thecAMP-binding protein Epac, J. Biol. Chem. 287 (2012) 44192-44202).

Assay of In Vitro GEF Activity:

In vitro GDP exchange catalyzed by Epac was measured using purifiedrecombinant Epac isoforms and Rap1A loaded with Bodipy-GDP, as describedin D. Courilleau, et al (above mentioned). The GEF activity of Epacproteins was initiated by injection of the agonist. Stock solutions ofCE3F4 and its analogs (12 mM in 100% DMSO) were kept at −20° C. and werediluted in assay buffer (0.67% final DMSO concentration) just before usefor GDP exchange assays. The release of Bodipy-GDP was measured in realtime as the decay of fluorescence. A single exponential was fit to thetime-course data using the Graphpad Prism program. The initial velocity(Vi) of GDP exchange on Rap1 was calculated as described in D.Courilleau et al.).

Statistical Analysis Examples 1 to 3

All data are expressed as means±standard error of the mean. Differencesin quantitative variables were examined by one-way analysis of variance(ANOVA) or paired two-tailed t test. p value<0.05 (*), p value<0.01 (**)and p value<0.001 (***). All analyses were performed using GraphPadPrism.

Examples 4 and 5

Data are expressed as mean±S.D. Differences in quantitative variableswere examined by unpaired one- or two-tailed Student's t test. EC₅₀ andIC₅₀ values were computed according to a four-parameters dose-responsemodel and compared on the basis of the extra sum-of square F test, usingGraphpad Prism. Two-tailed Wilcoxon matched-pair signed rank tests wereperformed using GraphPad Prism.

1-15. (canceled)
 16. A compound having the following formula:


17. (canceled) 